Cell communication through gap junctions occurs in most epithelial tissues of vertebrate organisms. It is thought to provide a route by which signals controlling growth, differentiation, and metabolic response to external signals are exchanged among cells of a communicating unit, permitting synchronous regulation despite differences in access to blood supply and innervation. To understand the potential of cells in the kidney to modulate their communication behavior, we have developed a cell culture system in which two kidney-derived cell lines (MDCK and HaK) can be shown to share potassium ions or uridine derivatives when in contact with each other. Preliminary studies indicate that exposure to ouabain inhibits both forms of communication in a concentration-dependent manner and that the mineralocorticoid hormone aldosterone counteracts the inhibition. These studies were all carried out in medium containing 10% fetal calf serum and using plastic or glass substrates for cell attachment. To provide a more reproducible and physiologically appropriate environment to study these phenomena, we now propose to develop a serum-free medium in which to perform the assays and to culture the cells on permeable membranes. Once these alterations in our culture procedure have been validated, we will compare new procedures with previous ones to see whether modulation of junctional communication by aldosterone is still apparent. Control studies will include tests of the competitive inhibitor spironolactone and the glucocorticoid dexamethasone. We will also develop a system in which the cells are cultured on small beads of various types in order to adapt fluorescence measurements designed for cells in suspension to the study of changes in cell calcium and pH that may accompany modulation of communication in these anchorage-dependent cells. The results will provide a model system for regulation of kidney cell communication that can then be used to approach questions of biochemical and physiological mechanism.